Reversal of cancer phenotype by inhibiting expression of prostate tumor inducing gene

ABSTRACT

This invention provides a method for reversing cancer phenotype of a cancer cell which comprises introducing an exogenous material which is capable of specifically recognizing either a Prostate Tumor Inducing Gene, RNA of said gene or gene product of said gene into the cell under conditions permitting inhibition of the expression of said gene or function of said gene product so as to thereby reverse the cancerous phenotype of the cell. This invention also provides a method for reversing cancer phenotype of a cancer cell in a subject which comprises introducing an exogenous material which is capable of specifically recognizing a Prostate Tumor Inducing Gene, RNA of said gene or gene product of said gene into the subject&#39;s cancer cell under conditions permitting inhibition of the expression of said gene or function of said gene product in the subject&#39;s cell so as to thereby reverse the cancerous phenotype of the cell.

This application is a Continuation-In-Part application of U.S. Ser. No.08/708,206, filed Sep. 6, 1996, the content of which is incorporatedinto this application by reference.

The invention disclosed herein was made with Government support underGrant No. NIH CA 35675. Accordingly, the U.S. Government has certainrights in this invention.

BACKGROUND OF THE INVENTION

Cancer of the prostate is a major clinical problem with the diagnosis of244,000 new cases and more than 40,500 deaths of American men predictedby the American Cancer Society in 1995. Currently, the predictedlifetime incidence of prostate cancer is 15% and the estimated lifetimedeath risk from this disease is approximately 3.4%. It is not possibleby present technologies to distinguish between cancers that will becomeclinically aggressive versus indolent cancers that will remainclinically benign. Current treatment protocols, including hormonaltherapy, radiation therapy and surgery have limitations. Hormonaltherapy requires a hormone-responsive tumor; when a tumor developshormone-insensitivity it can progress unchecked. Attempts at cure usingradiation therapy and surgery are limited to eradication of the primarytumors. However, tumor can escape surgical or radiotherapeutic ablation,and these approaches cannot be used to successfully cure, and rarelyeven to limit metastatic disease. In addition, even when successful,these approaches can significantly diminish the patient's quality oflife. These findings emphasize the need for improved diagnostic andtherapeutic approaches for identifying prostate carcinomas, predictingclinical aggressiveness and effectively treating patients with thiscancer.

Identifying the genetic elements mediating prostate cancer developmentand progression will lead to improved diagnostic tests and mayultimately result in gene-, immunological- and drug-based technologieswith therapeutic applications. Transfection of human prostate carcinoma(LNCaP) DNA into a new DNA acceptor cell line, CREF-Trans 6, andinjection into nude mice results in tumor formation (Su et al.,Anticancer Res. 12:297-304, 1992). Using tumor-derived CREF-Trans 6cells and differential RNA display, the new putative oncogene, prostatetumor inducing gene-1 (PTI-1), has been identified (Shen et al., PNAS92: 6788-6782, 1995). PTI-1 encodes a mutated and truncated humanelongation factor-1α (EF-1α). Normal EF-1α plays a prominent role inprotein translation, a process that is critical in controlling geneexpression and regulating cell growth. PTI-1 expression is observed inhuman prostate cancer cell lines (LNCaP, DU-145 and PC-3) andpatient-derived prostate carcinoma tissue samples (14 of 15), but not innormal prostate (6) or BPH (4) tissue. This observation suggests thatPTI-1 expression may be related specifically to carcinoma development.In addition, the observation that PTI-1 expression also occurs in a highproportion of carcinoma cell lines of the breast, colon and lungindicates that this genetic alteration may be a common event incarcinogenesis. If the modified EF-1α protein encoded by PTI-1 inhibitsthe ability of normal EF-1α to proofread mistakes in gene expressionthat mediate altered protein structure, then PTI-1 may function as amajor contributor to the mutator phenotype in specific human cancers.This putative aberrant processing resulting from PTI-1 expression hasbeen termed “translational infidelity”. If this hypothesis is validatedexperimentally, altered protein translation would represent a new andnovel mechanism underlying cancer development and progression. Targetedinhibition of PTI1, using genetic and/or drug interventional approaches,might therefore provide the basis for a novel strategy for the therapyof prostate cancer.

SUMMARY OF THE INVENTION

This invention provides a method for reversing cancer phenotype of acancer cell which comprises introducing a molecule capable ofspecifically recognizing a Prostate Tumor Inducing Gene into the cellunder conditions permitting inhibition of the expression of said gene soas to thereby reverse the cancer phenotype of the cell.

This invention also provides a method for reversing cancer phenotype ofa cancer cell in a subject which comprises introducing a moleculecapable of specifically recognizing a Prostate Tumor Inducing Gene intothe subject's cancer cell under conditions permitting inhibition of theexpression of said gene in the subject's cell so as to thereby reversethe cancer phenotype of the cell.

This invention provides a method for reversing cancer phenotype of acancer cell which comprises introducing a compound capable ofspecifically recognizing the RNA of a Prostate Tumor Inducing Gene intothe cell under conditions permitting inhibition of the expression ofsaid RNA so as to thereby reverse the cancer phenotype of the cell.

This invention also provides a method for reversing cancer phenotype ofa cancer cell in a subject which comprises introducing a compoundcapable of specifically recognizing the RNA of a Prostate Tumor InducingGene into the subject's cancer cell under conditions permittinginhibition of the expression of said RNA in the subject's cell so as tothereby reverse the cancer phenotype of the cell.

This invention provides a method for reversing cancer phenotype of acancer cell which comprises introducing a substance capable ofspecifically recognizing the gene product of a Prostate Tumor InducingGene into the cell under conditions permitting inhibition of thefunction of said gene product so as to thereby reverse the cancerousphenotype of the cell.

This invention provides a method for reversing cancer phenotype of acancer cell in a subject which comprises introducing a substance capableof specifically recognizing the gene product of a Prostate TumorInducing Gene into the subject's cancer cell under conditions permittinginhibition of the function of said gene product in the subject's cell soas to thereby reverse the cancer phenotype of the cell.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Stable expression of PTI-1 antisense reverses morphologicaltransformation. Stable transfection of CREF-Trans 6:4 NMT cells with apZeoSV construct (mediating resistance to Zeocin) containing thecomplete PTI-1 cDNA in an antisense orientation (pZeoSV-PTI-1-AS)results in colonies with a reverted flat morphology.

-   -   Cell Types:    -   1A CREF-Trans 6 (parental cell line, does not express PTI-1);    -   1B CREF-Trans 6:4 NMT (nude mouse tumor-derived clone of        CREF-Trans 6 cells transfected with high molecular weight LNCaP        human prostate cancer DNA, expresses PTI-1);    -   1C 4NMT-Vector (Zeocin resistant CREF-Trans 6:4 NMT clone        transfected with pZeoSV vector DNA);    -   1D 4NMT-PTI-1-AS cl 8 (flat variant of CREF-Trans 6:4 NMT cells        transfected with pZeoSV containing AS PTI-1).

FIG. 2. Stable expression of PTI-1 antisense suppresses anchorageindependent growth. A series of independent clones of CREF-Trans 6:4 NMTcells transfected with a pZeoSV construct containing the complete PTI-1cDNA in an antisense orientation. A total of 1×10⁵ cells in mediumcontaining 0.4% Noble agar was seeded on an agar medium containing 0.8%agar. Colonies were enumerated using an inverted tissue culturemicroscope after 21 days. The results are the average from 4 plates ±standard deviation from the mean. Cell types: CREF-Trans 6:4 NMT;4NMT-PTI-1-AS cl 1; 4NMT-PTI-1-AS cl 2; 4NMT-PTI-1-AS cl 5;4NMT-PTI-1-AS cl 6; 4NMT-PTI-1-AS cl 7; 4NMT-PTI-1-AS cl 8;4NMT-PTI-1-AS cl 9; 4NMT-PTI-1-AS cl 10; and 4NMT-PTI-1-AS cl 11.CREF-Trans 6 cells did not form colonies in agar (data not shown).

FIG. 3 Stable expression of PTI-1 antisense suppresses tumorigenesis.Confluent cultures were resuspended using trypsin-versene. Cells werewashed twice with serum free medium and viable cell counts determinedusing the trypan blue dye exclusion technique. Cells were resuspended inserum free medium at a concentration of 1×10⁷ cells/ml and 0.2 ml wasinjected subcutaneously into nude mice (Balb/c nu/nu, Taconic; 5 animalsper cell line.) Animals were monitored daily for tumor growth and tumorswere measured once a week with a caliper. Tumor volume was derived fromthe formula: π/6×larger diameter x (smaller diameter )². Cell types:CREF-Trans 6 (CREF); CREF-Trans 6:4 NMT (4 NMT); 4NMT-Vector (Vector);4NMT-PTI-1-AS cl 1 (cl 1); 4NMT-PTI-1-AS cl 6 (cl 6); 4NMT-PTI-1-AS cl 8(cl 8); and 4NMT-PTI-1-AS cl 10 (cl 10).

-   -   3A: Tumor data evaluated at the 14 day;    -   3B: Tumor data evaluated at the 21 day.

FIG. 4. Transcription of the 51 untranslated region (5′ UTR) of PTI-1occurs following induction of oncogenic transformation in rodent cellsand in specific human cancer cells. Nuclei from 2×10⁶ cells wereisolated form each of the cell lines shown and nuclear RNA was labeledin vitro and subsequently hybridized to denatured DNA probes onnitrocellulose filters. For each hybridization reaction, an equal numberof total counts representing a similar number of nuclei was used, sothat comparative rates of transcription could be obtained. Cell linedesignation:

-   -   4A: CREF (cloned rat embryo fibroblast); CREF-Ha-ras (CREF cells        transformed by the Ha-ras oncogene); CREF-HPV-51 (CREF cells        transformed by the E6/E7 transforming region of human papilloma        virus-51, CREF-v-src (CREF cells transformed by the v-src        oncogene)    -   4B: DU-145 (hormone refractive human prostate carcinoma cell        line); LNCaP (hormone responsive human prostate carcinoma cell        line); PC-3 (hormone retractive human prostate carcinoma cell        line);    -   4C: H0-1 (human melanoma cell line); MCF-7 (human breast        carcinoma cell line); T47D (human breast carcinoma cell line).        The gene probes utilized were: GAPDH (glyceraldehyde phosphate        dehydrogenase); 5′ UTR PTI-1 (5′ untranslated region of PTI-1):        pBR322 (bacterial DNA sequences).

FIG. 5. Induction of tumors in nude mice by PTI-1. Pooled zeocinresistant CREF-Trans 6 cells transfected with a pZeoSV vector or a 1.9kb PTI-1 S cDNA, a 5′ UTR PTI-1 S DNA or a Trun-EF PTI-1 S DNA clonedinto a pZeoSV vector were injected at 1×10⁶ cells per nude mouse (n=4).Animals were also injected with untransfected CREF-Trans 6 cells,CREF-Trans 6:4 NMT cells (NMT; tumor-derived CREF-Trans 6 cellstransfected with LNCaP high molecular weight DNA) and pooled CREF-Trans6 cells transfected with the combination of a separate 5′ UTR PTI-1 Sand a Trun-EF S DNA cloned into pZeoSV vectors. Tumors developed in themajority of 1.9 kb PTI-1 S cDNA and 4NMT injected animals by 7 days, andin all 1.9 kb PTI-1 S cDNA and 4NMT injected animals by 10 days.

FIG. 6. Expression of PTI-1 in nude mouse tumor-derived CREF-Trans 6cells. Tumors were isolated from animals, established in culture in thepresence of zeocin, total RNA was isolated and analyzed by Northernblotting. Northern blots were sequentially hybridized with a PTI-1,zeocin and then a GAPDH cDNA probe. NHPE, normal human prostateepithelial cells; PTI-1: NMT 1-1, 1-2, 2-2, 2-1, 3-1, 4-1 and 4-3represent independent tumors isolated from different animals.

FIG. 7. Inhibition of colony formation in PTI-1 expressing cells byantisense PT1-1. The indicated cell line was transfected with a pZeoSVplasmid containing or lacking an AS 0 to 500 PTI-1 5′ UTR, an AS Trun-EFregion of PTI-1 or an AS PTI-1 1.9 kb sequence. Transfected cells wereselected in zeocin and colony formation was determined. Results are themean using 4 test plates per experimental condition ±S.D. Similarresults were obtained ±10% in 2 additional experiments.

FIG. 8. Expression of AS PTI-1 and the absence of S PTI-1 inmorphologically reverted PTI-1 AS CREF-Trans 6:4 NMT clones. RNaseprotection assays using a 357 nt S PTI-1 probe demonstrate the presenceof PTI-1 AS transcripts in phenotypically reverted 4 NMT-PTI-1-ASexpressing CREF-Trans 6:4 NMT clones. RNase protection assays using a465 nt AS PTI-1 probe demonstrate the absence of PTI-1 S transcripts inphenotypically reverted 4 NMT-PTI-1-AS expressing CREF-Trans 6:4 NMTclones.

FIG. 9. Transcription of the 5′ UTR of PTI-1, EF-1α and GAPDH inCREF-Trans 6:4 NMT, 4NMT-Vector and 4NMT-PTI-1-AS cells. Nuclear run-onassays were performed using the indicated cell types. DNA probesimmobilized on filters include 5′ UTR (0 to 500 bp region of PTI-1),Trun-EF (EF-1α isolated from PTI-1), pBR322 and GAPDH.

DETAILED DESCRIPTION OF THE INVENTION

This invention provides a method for reversing cancer phenotype of acancer cell which comprises introducing a molecule capable ofspecifically recognizing a Prostate Tumor Inducing Gene into the cellunder conditions permitting inhibition of the expression of said gene soas to thereby reverse the cancerous phenotype of the cell.

This invention also provides a method for reversing cancer phenotype ofa cancer cell in a subject which comprises introducing a moleculecapable of specifically recognizing a Prostate Tumor Inducing Gene intothe subject's cancer cell under conditions permitting inhibition of theexpression of said gene in the subject's cell so as to thereby reversethe cancerous phenotype of the cell.

This invention provides a method for reversing cancer phenotype of acancer cell which comprises introducing a compound capable ofspecifically recognizing the RNA of a Prostate Tumor Inducing Gene intothe cell under conditions permitting inhibition of the expression ofsaid RNA so as to thereby reverse the cancerous phenotype of the cell.

This invention also provides a method for reversing cancer phenotype ofa cancer cell in a subject which comprises introducing a compoundcapable of specifically recognizing the RNA of a Prostate Tumor InducingGene into the subject's cancer cell under conditions permittinginhibition of the expression of said RNA in the subject's cell so as tothereby reverse the cancerous phenotype of the cell.

This invention provides a method for reversing cancer phenotype of acancer cell which comprises introducing a substance capable ofspecifically recognizing the gene product of a Prostate Tumor InducingGene into the cell under conditions permitting inhibition of thefunction of said gene product so as to thereby reverse the cancerousphenotype of the cell.

This invention provides a method for reversing cancer phenotype of acancer cell in a subject which comprises introducing a substance capableof specifically recognizing the gene product of a Prostate TumorInducing Gene into the subject's cancer cell under conditions permittinginhibition of the function of said gene product in the subject's cell soas to thereby reverse the cancerous phenotype of the cell.

In an embodiment of the above methods, the cancer cell is a prostatecancer cell, a breast cancer cell, a colon cancer cell, or a lung cancercell.

In another embodiment of the above methods, the Prostate Tumor InducingGene is PTI-1, PTI-2 or PTI-3. PTI-1, -2 and -3 are described incopending U.S. Ser. No. 08/371,377, filed Jan. 11, 1995 and PatentCooperation Treaty Application No. PCT/US96/00307, filed Janary 1996.

In an embodiment, the molecule capable of specifically recognizing aProstate Tumor Inducing Gene is a nucleic acid molecule. In anotherembodiment, the compound capable of specifically recognizing the RNA ofa Prostate Tumor Inducing Gene is a nucleic acid molecule. These nucleicacid molecules may be a ribozyme. These nucleic acid molecule maycomprise an expression vector. Specifically, these nucleic acid moleculemay have sequence complementary to the unique region of the ProstateTumor Inducing Gene. Such region may be the 5 prime or the 3 primeuntranslated region of the PTIs. These nucleic acid molecules may alsobe complementary to the untranslated region of the PTIs.

In another embodiment, the nucleic acid molecule is complementary to the5′ untranslated region and the EF-1α region of the said gene. In afurther embodiment, the nucleic acid molecule comprises the sequence of5′ AAATTAAGCTATGCAGTCGG3′.

In a further embodiment, the nucleic acid comprises a vector. The vectorincludes, but is not limited to, an adenovirus vector, adeno-associatedvirus vector, Epstein-Barr virus vector, Herpes virus vector, attenuatedHIV virus, retrovirus vector and vaccinia virus vector.

Methods to introduce a nucleic acid molecule into cells have been wellknown in the art. Naked nucleic acid molecule may be introduced into thecell by direct transformation. Alternatively, the nucleic acid moleculemay be embedded in liposomes. Accordingly, this invention provides theabove methods wherein the nucleic acid is introduced into the cells bynaked DNA technology, adenovirus vector, adeno-associated virus vector,Epstein-Barr virus vector, Herpes virus vector, attenuated HIV vector,retroviral vectors, vaccinia virus vector, liposomes, antibody-coatedliposomes, mechanical or electrical means. The above recited methods aremerely served as examples for feasible means of introduction of thenucleic acid into cells. Other methods known may be also be used in thisinvention.

In another embodiment, the substance capable of specifically recognizingthe gene product of a Prostate Tumor Inducing Gene is an antibody whichspecifically binds to the protein of a Prostate Tumor Inducing Gene.

This invention provides a method for killing a cancer cell whichcomprises introducing the promoter of a Prostate Tumor Inducing Geneoperatively linked to nucleic acid having sequence which encodes atleast one toxic factor into the cell under conditions permittingexpression of said toxic factor so as to thereby kill the cancer cell.

This invention also provides a method for killing a cancer cell in asubject which comprises introducing the promoter of a Prostate TumorInducing Gene operatively linked to nucleic acid having sequence whichencodes at least one toxic factor into the subject's cancer cell underconditions permitting expression of said toxic factor in the subject'scell so as to thereby kill the cancer cell.

This invention provides a pharmaceutical composition which comprises anamount of a molecule capable of specifically recognizing a ProstateTumor Inducing Gene effective to inhibit the expression of the ProstateTumor Inducing Gene, thereby reversing the cancer phenotype of a cancercell and a pharmaceutically acceptable carrier.

As used herein, the term “pharmaceutically acceptable carrier”encompasses any of the standard pharmaceutical carriers. Thepharmaceutical composition may be constituted into any form suitable forthe mode of administration selected. Compositions suitable for oraladministration include solid forms, such as pills, capsules, granules,tablets, and powders, and liquid forms, such as solutions, syrups,elixirs, and suspensions. Forms useful for parenteral administrationinclude sterile solutions, emulsions, and suspensions.

This invention provides a pharmaceutical composition which comprises anamount of a compound capable of specifically recognizing the RNA of aProstate Tumor Inducing Gene effective to inhibit the expression of theRNA of the said gene, thereby reversing the cancer phenotype of a cancercell and a pharmaceutically acceptable carrier.

This invention provides a pharmaceutical composition which comprises anamount of a substance capable of specifically recognizing the geneproduct of a Prostate Tumor Inducing Gene effective to inhibit thefunction of such gene product, thereby reversing the cancer phenotype ofa cancer cell and a pharmaceutically acceptable carrier.

This invention also provides a pharmaceutical composition comprising anamount of a nucleic acid molecule which is complementary to a ProstateTumor Inducing Gene effective to inhibit the expression of said gene anda pharmaceutically acceptable carrier.

This invention provides a pharmaceutical composition comprising anamount of a nucleic acid molecule which is complementary to the 5′untranslated region and the EF-1α region of a Prostate Tumor InducingGene effective to inhibit the expression of said gene and apharmaceutically acceptable carrier. In an embodiment of thepharmaceutical composition, the nucleic acid molecule comprises thesequence of 5′ AAATTAAGCTATGCAGTCGG3′.

This invention provides the above pharmaceutical composition, whereinthe cancer cell is a prostate cancer cell, a breast cancer cell, a coloncancer cell, or a lung cancer cell.

Finally, this invention provides a method for reversing cancer phenotypeof a cancer cell in a subject which comprises administering the abovepharmaceutical composition into the subject so as to reverse the cancerphenotype of the cell.

This invention will be better understood from the Experimental Detailswhich follow. However, one skilled in the art will readily appreciatethat the specific methods and results discussed are merely illustrativeof the invention as described more fully in the claims which followthereafter.

Experimental Details

The ability of PTI-1 to discriminate between normal prostate, BPH andprostate carcinoma suggests that PTI-1 may prove valuable as adiagnostic and a therapeutic reagent for prostate cancer. In preliminarystudies, the potential use of PTI-1 for detecting prostate carcinomacells diluted with normal cells and the presence of metastatic prostatecarcinoma cells in the peripheral circulation has been evaluated. PTI-1expressing LNCaP cells were serially diluted with PTI-1 nonexpressingCREF-Trans 6 cells, cellular RNAs were isolated and analyzed by RT-PCRusing primers specific for the unique 5′ region of PTI-1. Additionally,the same RNA samples were analyzed by RT-PCR using primers specific fortwo well-established prostate gene products, PSA and PSM. Using thisapproach, PTI-1 could detect the equivalent of one LNCaP cell diluted in10⁸ CREF-Trans 6 cells, whereas the sensitivity of PSA and PSM maximallydetected one LNCaP cell diluted in 10⁶ CREF-Trans 6 cells. This findingis important since recent studies indicate that the detection ofblood-borne PSA-synthesizing cells by RT-PCR can be accomplished inpatients with localized as well as metastatic prostate cancer and thatthis detection provides a reliable marker for predicting local invasionof a prostate tumor prior to surgical procedures. In preliminarystudies, several blood RNA samples from patients with metastatic diseasewere analyzed by RT-PCR and found to be positive for both PSA and PTI-1.The biological significance of the prostate cells in the circulation isnot presently known. However, the high percentage of frank metastaticpatients that give a positive reaction on this assay suggests that it isdetecting circulating metastatic prostate cells. Multiple mRNA tissueNorthern blots (Clontech) have now been used to determine which normalhuman tissue contains mRNA that hybridizes with the 5′ unique region ofPTI-1 and the EF-1α region of PTI-1.

These experiments indicate that the unique 5′ region of PTI-1 isexpressed in skeletal muscle and in colon, but not in spleen, thymus,prostate, testis, ovary, small intestine, peripheral blood, heart,brain, placenta, lung, liver, kidney or pancreas. In contrast, probingthe same Northern blots with a region of the EF-1α region of PTI-1results in expression in all of the tissues tested. PTI-1 expression hasalso been detected using RT-PCR in blood RNA samples from patients withmetastatic disease that have also been found to be positive for PSAexpression. These data, which indicate that PTI-1 is 100× more sensitivethan PSA or PSM in detecting prostate carcinoma cells, that blood RNAsfrom patients with metastatic prostate disease are positive for both PSAand PTI-1 and that peripheral blood cells do not express PTI-1, suggestthat PTI-1 may prove selective and sensitive for the early detection ofprostate carcinoma cells in the peripheral circulation.

The ability of anti-sense PTI-1 (PTI-1-AS) to alter the transformedphenotype of tumor derived LNCaP DNA transfected CREF-Trans 6(CREF-Trans 6:4 NMT) cells has been determined (FIG. 1-3). PTI-1 wascloned in an antisense orientation (PTI-1-AS) into pZeoSV vector, thatcontains a cloning site adjacent to a SV40 enhancer and promoter andalso contains a selectable Zeocin resistance gene controlled by a CMVpromoter. Transfection of this construct into CREF-Trans 6:4 NMTfollowed by growth in Zeocin resulted in the identification of severalZeocin resistant colonies displaying a reverted normal CREF-Trans 6-likemorphology (FIG. 1). These PTI-1-AS-reverted clones were isolated andfound to exhibit a reduced ability to form colonies when seeded in agar,i.e., ˜40% for parental CREF-Trans 6:4 NMT cells versus ˜≦10% for thedifferent PTI-1-AS-reverted clones (FIG. 2). This observation indicatesthat altering PTI-1 expression may mediate a reversal in the transformedproperties of PTI-1-expressing cells. When anti-sense PTI-1 expressingrevertant, CREF-trans 6:4 NMT cells are injected into nude mice, tumorformation is prevented (FIG. 3A and 3B). These exciting findings clearlywarrant further studies. These include determining the effect ofblocking PTI-1 expression on tumorigenic properties of CREF-Trans 6:4NMT cells and determining the effect of PTI-1-AS expression, withphosphorothioate oligonucleotides, additional expression vectorconstructs and adenoviral constructs, on the in vitro and in vivoproperties of human prostate cancer cells, such as LNCaP, DU-145 andPC-3 cells. The efficacy of adenoviral vectors for reversing prostatecancer by constructing vectors expressing the anti-sense RNA of PTI-1will be important to test. The efficacy of adenovirus vectors containinga PTI-1-AS gene in reversing the in vitro transformation-related and invivo oncogenic properties of human prostate cancer cells in nude micexenograft experiments also requires testing. The continued success ofthis anti-sense approach in reversing the cancer phenotype would serveas the basis for expanded in vivo animal studies. These types of studieswill be necessary as a first step in validating PTI-1-AS for therapeuticapplications in males with prostate cancer.

The full-length PTI-1 cDNA is 2,123 bp and consists of a unique 630 bp5′ region and a 3′ region extending from 630 to 2,123 bp that displays97% homology to a truncated and mutated (6 point mutations) human EF-1αgene. Since PTI-1 encodes a truncated version of EF1α, 46 kDa versus 50kDa for wild-type human EF1α, antibodies produced against specificregions of the EF-1α protein may prove amenable for detecting PTI-1expression in cancer tissue. This possibility will be determined byimmunoprecipitation and Western blotting analysis with peptide-derivedrabbit polyclonal antibodies prepared against the N-terminus of humanEF-1α (missing in the PTI-1 gene), antibodies prepared against thespecific mutated regions of EF-1α present in PTI-1 and antibodiesprepared against the C-terminus of human EF-1α. If carcinoma cellscontain both EF-1α and PTI-1, then N-terminus antibodies should reactonly with the ˜50 kDa EF-1α protein whereas C-terminus antibodies shouldreact with both the ˜50 kDa EF-1α and the ˜46 kDa PTI-1 proteins. Inthis context, detection of the ˜46 kDa PTI-1 protein could provediagnostic for carcinomas.

PTI-1 also contains six point mutations in its EF-1α gene region thatallows it to be distinguished from wild-type human EF-1α. At the presenttime, the significance of these specific point mutations in the EF-1αcoding region of PTI-1 is not known. To determine the frequency of thespecific mutations in EF-1α during human prostate carcinoma developmentthe single-stranded conformation polymorphism (SSCP) technique will beused. A single nucleotide difference between two short single-strandedDNA molecules can produce a difference in the conformations sufficientto produce changes in the molecules' electrophoretic mobilities onneutral polyacrylamide gels. Using SSCP it will be possible to determinethe frequency of specific mutations in the EF-1α coding region of PTI-1in human prostate cancers. Sequence analyses will be used to confirm themutational changes.

Preliminary experiments indicate that PTI-1 expression can be detectedin RNA samples isolated from prostate carcinoma cell lines,patient-derived prostate carcinoma tissue samples and blood samples frompatients with metastatic disease. These studies are being expanded usinga minimum of 30 histologically confirmed samples from normal prostate(autopsy), patient-derived BPH and patient-derived prostate carcinoma.Blood RNA samples from patients with and without confirmed metastaticdisease and with and without confirmed PSA and PSM expression will alsobe evaluated for expression of PTI-1. These studies will be necessary tostatistically confirm the initial observation of differential expressionof PTI-1 in human prostate carcinomas versus normal prostate and BPH andexpression of PTI-1 in blood samples from patients with metastaticdisease. To define the precise localization within the prostate of PTI-1expression we will use in situ hybridization. Coexpression of PSA willbe determined by immunohistochemistry. Identification of the specificcell types expressing PTI-1 in prostate tumors will permit investigationof the role PTI-1 expression plays in the neoplastic transformation andtumor progression in the prostate carcinoma. Analysis of expression innormal, benign hyperplastic and neoplastic human prostate will allowcomparison of expression in intraepithelial, non-invasive neoplasms andin cancers that show histologic progression from low-grade andhigh-grade invasive tumors, even in a single patient. For this studyboth fresh frozen and paraffin-embedded tissue will be used. Seriallycut tissue sections will be prepared for both in situ analyses androutine hematoxylin and eosin histologic analysis so that an accuratepathologic assessment of the prostate disease in each microscopic fieldcan be made and appropriate correlation of tumor progression withexpression data inferred. This is especially important in prostate wherea single tissue section can show fields ranging from benign glands, tointraepithelial non-invasive neoplasia, to invasive cancer of low,intermediate and high grades. The Gleason's grading system will be usedto assess the degree of differentiation in invasive prostateadenocarcinoma.

The above findings indicate that (1) PTI-1 expression is restricted tomale neoplastic tissue, (2) PTI-1 is a more sensitive indicator than PSAor PSM for detecting prostate carcinoma cells and (3) inhibiting PTI-1expression by using PTI-1-AS can reverse the transformation phenotype.

Second Series of Experiments

The PTI-1 gene has been identified as a putative oncogene using DNAtransfection and nude mouse tumor assays. By comparing RNAs synthesizedin untransfected and tumor-derived human LNCaP prostate carcinomaDNA-transfected CREF-Trans 6 cells, a novel 214-bp sequence wasidentified using differential RNA display that is expressed in LNCaP andLNCaP DNA-transfected tumor-derived CREF-Trans 6 cells. By screening anLNCaP cDNA library and using the 5′-RACE procedure, a full-length 2.0-kbPTI-1 cDNA was obtained. Sequence analysis indicates that the PTI-1 genecontains a 630-bp 5′ sequence (with ˜85% sequence homology to Mycoplasmahyodneumoniae 23S ribosomal RNA) and a 3′ sequence homologous to atruncated and mutated form of human elongation factor 1α (EF-1α).Probing Northern blots with a DNA fragment corresponding to the 5′region of PTI-1 identifies multiple PTI-1 transcripts in RNAs from humancarcinoma cell lines derived from the prostate, lung, breast and colon.By using a pair of primers recognizing a 280-bp region within the 630-bp5′ untranslated region (UTR) of PTI-1, reverse transcription-PCR detectsPTI-1 expression in patient-derived prostatic carcinomas but not innormal prostate or BPH tissue. In contrast, reverse transcription-PCRdetects prostate-specific antigen expression in all of the prostatetissue. These results indicate that PTI-1 may be a member of a class ofoncogenes that could affect protein translation and contribute tocarcinoma development in human prostate and other tissues.

Aims: To evaluate the diagnostic and therapeutic potential of a putativedominant-acting nude mouse prostatic carcinoma tumor-inducing gene,PTI-1.

A. Characterizing the PTI-1 Gene and Defining its Mode of Action.

The homology between the 5′ UTR of PTI-1 and bacterial ribosomal 23S RNAmandated that experiments be performed to confirm the authenticity ofthe PTI-1 cDNA. In particular, it was important to rule out thepossibility that PTI-1 resulted from a cloning artifact or mycoplasmalcontamination. To approach these questions a PCR strategy was employedusing primer sequences located in the 5′ UTR and in the EF-1α region ofPTI-1. RNAs were isolated from cell lines previously shown to bepositive by both Northern blotting and RT-PCR using probes generatedwithin the 5′ UTR region of PTI-1. Using this approach, PCR fragments ofthe predicted size were obtained using RNA isolated from CREF-Trans 6:4NMT and DU-145. Sequence analysis of the PCR amplified DNAs indicatedthe presence of both the 5′ UTR and 3′ EF-1α region indicating theauthenticity of the PTI-1 cDNA. Since the PTI-1 cDNA isolated directlyfrom the LNCaP cDNA library consists of 1.8-kb, primers within the200-bp region of the 5′ UTR missing in the PTI-1 cDNA were used for PCRamplification. Using this region, amplified fragments with theappropriate size and sequence were obtained from cells found to bepositive using 5′ UTR sequences within the cDNA of PTI-1 and the bridgeregion (5′ UTR and 3′ EF-1α sequences) of PTI-1.

It was important to determine if the 5′ UTR region of PTI-1 is presentin the human genome. To address this question, genomic DNA was isolatedfrom human kidney tissue and PCR amplification was performed usingprimers within the 5′ UTR region of PTI-1. With this approach, a band ofthe appropriate size and sequence was obtained indicating that this 5′UTR sequence is part of the human genome. Similarly, analyses of DNAsisolated from different species, including bacteria, yeast, monkey, ratand human, indicate the presence of PTI-1 5′ UTR sequences. The abilityto utilize PTI-1 for diagnostics and therapy will depend on preferentialexpression in human cancers. To determine the spectrum of tissueexpression, RNAs have been isolated from normal human cerebral cortex,cerebellum, liver, kidney and testis and found to be negative for PTI-1expression.

These studies indicate: (1) PTI-1 is an authentic cDNA expressed invarious human cancer cells; (2) The 5′ UTR of PTI-1, with sequencehomology to bacterial ribosomal 23S RNA, is found in the genome of botheukaryotes and prokaryotes; and (3) The 5′ UTR of PTI-1 is not expressedin a spectrum of normal tissue.

B. Determining the Effect of Antisense PTI-1 Expression on theBiological Properties of PTI-1 Expressing Transformed and TumorigenicCells.

Antisense technology represents a potentially useful strategy forselectively inactivating specific target genes. To determine ifsuppression of PTI-1 expression could modify the transformed phenotypein LNCaP DNA-transfected nude mouse tumor-derived CREF-Trans 6 cells,CREF-Trans 6:4 NMT, antisense PTI-1 constructs were prepared in a Zeocinexpression vector system. Transfection of CREF-Trans 6:4 NMT cells witha Zeocin-PTI-1 (AS) construct, but not with a Zeocin vector lacking theantisense PTI gene, and selection in Zeocin resulted in Zeo_(R) colonieswith a flat morphology (FIG. 1). Morphologically reverted PTI-1 (AS)containing CREF-Trans 6:4 NMT cells also displayed a suppression inanchorage independent growth. (FIG. 2). Studies have been conducted todetermine the in vivo tumorigenic properties in nude mice of PTI-1 (AS)containing CREF-Trans 6:4 NMT cells. When four anti-sense transfectedflat revertant of CREF trans 6:4 NMT cells are injected into a thymicnude mice, tumor formation is prevented (FIG. 3A & 3B). Additionally,the Zeocin PTI-1 (AS) construct has been transfected into DU-145 cellsto determine its effect on in vitro transformation and in vivooncogenicity in nude mice.

The above studies suggest that PTI-1 (AS) can effectively revert thetransformed phenotype of cells expressing PTI-1. These observationssuggest that further studies using adenovirus vectors, for genedelivery, are warranted. The use of adenovirus vectors to expresstherapeutic gene products has great potential, especially if theexpression of the desired gene is only required transiently to suppressmalignant cell growth, so that the natural defense mechanisms of thepatient can gain the upper hand. Accordingly adenovirus vectors arebeing constructed to express RNAs corresponding to antisense sequencesof PTI-1. The antisense version should suppress the oncogenic phenotypeof CREF-Trans 6:4 NMT and human prostate carcinoma cells. The techniqueemployed to create the adenovirus vectors is essentially that of Grahamand his colleagues, in which a small bacterial plasmid, containing theleft hand end of the adenovirus genome, is modified to encode the geneof choice under the control of a suitable mammalian promoter; withappropriate RNA processing signals also included. The cassette is thenincorporated into the full length adenovirus genome using homologousrecombination between the cassette plasmid and a cloned adenovirusgenome, after co-transfection into human cells. Followingco-transfection, the recombinant virus is plaque-purified and examinedfor expression of the desired gene product. Homologous recombination isthe rate-limiting step in this method, and so we have modified thecassette plasmid to increase the available homology between it ant thefull length genome. This has led to a considerably increased successrate in the construction of a range of vectors. The antisense versionsof the whole PTI-1 gene and the 5′ UTR region of this gene are beingcreated. These vectors available will therefore be available foranalysis soon.

C. Analyze PTI-1 Expression Using in situ Hybridization Approaches inProstate Carcinomas of Differing Decrees of Differentiation and HormoneResponsiveness Versus Prostatic Intraerithelial Neoplasia (PIN), BenignProstatic Hypertrophy (BPH) and Normal Prostate Tissues.

A variety of optimally handled tissues suitable for expression analysishas now been obtained. These include samples of normal and hyperplasticprostate, in addition to prostate carcinomas exhibiting a range ofdifferentiation varying from in situ, non-invasive carcinoma (PIN), tooinvasive well differentiated and invasive poorly differentiated tumors.The PTI-1 cDNA has been cloned into vectors suitable for riboprobesynthesis and in situ hybridization analysis. Probes prepared includeportions of the PTI-1 5′ UTR, in addition to the “bridge region”spanning the 5′ UTR junction with the EF-1α homologous sequence. SensePTI-1 and antisense PSA riboprobes are being used as experimentalcontrols. Using these approaches initial experiments are under way todefine the stages of transformation in prostate carcinoma with whichPTI-1 expression is associated.

D. Optimizing the Use of PTI-1 as a Probe for Detecting ProstateCarcinoma Cells in the Peripheral Circulation of Patients.

Confirmation of the authenticity of PTI-1 by demonstrating the presenceof the bridge region (5′ UTR linked to EF-1α) was a priority for furthertranslational studies on this interesting gene. Experiments have beenperformed to directly determine the relationship between PSA, PSM andPTI-1 expression using RT-PCR with RNAs isolated from peripheral bloodfrom patients with and without confirmed metastatic carcinoma of theprostate. These studies documented that PTI-1 is more efficient indetecting patients with presumably metastatic prostatic carcinoma cellin the blood stream than either PSA or PSM (Sun, Y., Lin, J., Katz, A.E. & Fisher, P. B. (1997) Cancer Res. 57, 18-23.).

Third Series of Experiments

Differential Effect of PTI-1 Antisense Expression on Colons Formation inNormal Cloned Rat Embryo fibroblast (CREF-Trans 6), Human ProstateCarcinoma DNA-transformed CREF-Trans 6 and Ha-ras Oncogene-transformedCREF Cells.

To determine if the effect of PTI-1 antisense [PTI-1 (AS)] is specificfor cells expressing the PTI-1 gene or the 5′ untranslated region of thePTI-1 gene (5′ UTR) lipofectin-mediated transfection studies have beenperformed. The cell types used included CREF-Trans 6 (negative for PTI-1expression), CREF-Trans 6:4 NMT (tumor-derived CREF-Trans 6 cellstransfected with high molecular weight DNA from LNCaP human prostatecancer cells) (positive for PTI-1 expression) and CREF-ras (CREF cellstransformed by the Ha-ras oncogene) (positive for expression of theunique 5′ UTR of PTI-1).

Experimental Procedure: CREF-Trans 6, CREF-Trans 6:4 NMT and CREF-rascells were seeded at 1×10⁶ per 60 mm-plate and 24 hr later cultures weretransfected using 50 mg per plate of lipofectin with 10 ug of pZeoSVvector DNA (conferring resistance to Zeocin) or pZeoSV-PTI-1 (AS)construct DNA (containing the PTI-1 gene in an antisense orientation inthe pZeoSV vector). Forty-eight hr later, transfected cells werereseeded at 1×10⁵ cells/60-mm plate and the next day the medium waschanged with the addition of Zeocin (50 to 100 ug/ml). Cultures wererefed with Zeocin-containing medium every 3 to 4 days and colonies werefixed and stained with Giemsa after 3 weeks culture. The average numberof Zeocin-resistant colonies forming in 4 plates was determined and theratio between vector and PTI-1 (AS) colony formation was determined.

Results: Ratio Cell Type pZeoSV (Vector) pZeoSV-PTI-1 (AS) V/ASCREF-Trans 6 71 ± 7 66 ± 7 1.1 CREF-Trans 6:4 NMT 68 ± 7 30 ± 4 2.3*CREF-ras 37 ± 7 17 ± 3 2.2**Significant reduction in colony formation in PTI-1 (AS) transfectedversus pZeoSV transfected cultures.

Conclusion: Antisense PTI-1 can suppress colony formation in transformedand tumorigenic CREF-Trans 6 cells, whereas this effect is not observedin normal CREF-Trans 6 cells. Since CREF-Trans 6:4 NMT expresses thecomplete PTI-1 gene and CREF-ras only expresses the unique 5′ UTR ofPTI-1, inhibition of either a partial region of PTI-1 (5′ UTR) or thecomplete PTI-1 gene using antisense technology can inhibit the growthand colony formation of tumorigenic transformed cells.

Fourth Series of Experiments

Transcription of the 5′ Untranslated Region (5′ UTR) of PTI-1 inTransformed Rodent and Human Cancer Cells

Background: PTI-1 expression is detected using Northern blotting inhuman prostate carcinoma cell lines, human prostate carcinomas frompatients and specific human -carcinoma cell lines derived from thebreast (T47D), colon (SW480 and LS174T) and lung (NCI H-69). Incontrast, PTI-1 is not expressed in normal prostate tissue, benignprostate hypertrophy tissue, specific human cancer cell lines (such asthe human melanoma cell line H0-1 and the specific strains of humanbreast carcinoma cell line MCF-7), various normal cell lines andCREF-Trans 6 cells. The ability to be expressed as mRNA suggests thatthe PTI-1 gene is being transcribed by the appropriate cell type.Studies have been conducted to determine if the 5′ UTR of PTI-1 isexpressed in additional cell types and if transformation by diverseoncogenes activates transcription of the 5′ UTR of PTI-1.

Experimental Protocol: The transcription of the unique 5′ UTR of PTI-1was determined using nuclear run-on assays as previously described (1).Nuclei from approximately 2×10⁶ cells were isolated and RNA previouslyinitiated by RNA polymerase II were allowed to elongate in the presenceof [³²P]UTP. Nuclear RNA was isolated, purified by filtering through aG-50 Sephadex column. Nuclear RNA was extracted, purified by filteringonto Millipore (0.45-um) filters followed by elution and denaturing bytreatment with 0.1 M sodium hydroxide for 5 min on ice. Nylon membranescontaining 2 μg of the appropriate denatured plasmid DNA gene insertwere hybridized with [³²P]-labeled RNA from the different cell types asdescribed (2,3).

Cell Types Include:

-   CREF (cloned rat embryo fibroblast),-   CREF-Ha-ras (CREF cells transformed by the Ha-ras oncogene),-   CREF-HPV-51 (CREF cells transformed by the human papilloma virus-51    E6/E7 transforming genes)-   CREF-V-src (CREF cells transformed by the V-src oncogene)-   DU-145 (hormone refractive human prostate carcinoma cell line)-   LNCaP (hormone responsive human prostate carcinoma cell line)-   PC-3 (hormone refractive human prostate carcinoma cell line)-   H0-1 (human melanoma cell line)-   T47D (human breast carcinoma cell line)

DNA Probes Include:

-   GAPDH-(glyceraldehyde phosphate dehydrogenase)-   5′ UTR-PTI-1 (5′ UTR region of the PTI-1 gene)-   pBR322 (bacterial negative control gene sequences)

Results: CREF cells contain transcripts for GAPDH, but not the 5′ UTR ofPTI-1 or pBR322. CREF cells transformed by the Ha-ras oncogene(CREF-Ha-ras), HPV-51 (CREF-HPV-51) and V-src (CREF-V-src) transcribethe GAPDH and the PTI-1 5′ UTR sequences. All three prostate carcinomacell lines, DU-145, LNCaP and PC-3, express GAPDH and the 5′ UTR ofPTI-1. The H0-1 human melanoma and the MCF-7 human breast carcinoma celllines do not transcribe the 5′ UTR of PTI-1. The human T47D breastcarcinoma cell line contains transcripts for the 5′ UTR of PTI-1.

Conclusion: Transformation of CREF cells by diverse oncogenes, thatresult in morphological transformation and acquisition of oncogenicpotential, result in the activation of the 5′ UTR of PTI-1. Theseresults implicate expression of genes linked to this sequence ascomponents of the transformation process. This sequence may thereforeserve as a direct target for inactivation and reversal of the cancerphenotype. The 5′ UTR of PTI-1 is transcriptionally active in prostatecarcinoma and breast carcinoma cells containing mRNA for the PTI-1 gene.In contrast, no transcription or expression on a Northern level isapparent in H0-1 and T47D human cancer cells. These results demonstratethat expression of PTI-1 (both the 5′ UTR region and the complete PTI-1mRNA) is restricted to specific cancer cells, including 100% of prostatecarcinoma cell lines and specific breast carcinoma cell lines). In thiscontext, targeting the inactivation of the 5′ UTR of PTI-1 and thefull-length PTI-1 gene may prove useful for reversing the cancerphenotype. The absence of expression of this gene in normal cellssuggests that antisense, and similar strategies, designed to inactivatethe PTI-1 gene may prove useful for cancer therapy.

Refs.

-   1. Su, Z. -z., Yemul, S., Estabrook, A., Friedman, R. M., Zimmer, S.    G., and Fisher, P. B. Transcriptional switching model for the    regulation of tumorigenesis and metastasis by the Ha-ras oncogene:    transcriptional changes in the Ha-ras tumor suppressor gene lysyl    oxidase. Intl. J. Oncol., 7: 1279-1284, 1995.-   2. Su, Z. -z., Austin, V. N., Zimmer, S. G., and Fisher, P. B.    Defining the critical gene expression changes associated with    expression and suppression of the tumorigenic and metastatic    phenotype in Ha-ras-transformed cloned rat embryo fibroblast cells.    Oncogene, 8: 1211-1219, 1993.-   3. Jiang, H., Waxman, S., and Fisher, P. B. Regulation of c-fos,    c-jun and jun-B gene expression in human melanoma cells induced to    terminally differentiate. Mol. Cell. Different., 1: 197-214. 1993.    Fifth Series of Experiments

The genetic alterations and molecular events mediating human prostatecancer development and progression remain to be defined. Rapidexpression cloning and differential RNA display detect a novel putativeoncogene, prostate tumor inducing gene-1 (PTI-1), that is differentiallyexpressed in human prostate (as well as breast, colon and small celllung) cancer cell lines, patient-derived prostate carcinomas and bloodfrom patients with metastatic prostate cancer. PTI-1 consists of aunique 5′ untranslated region (5′ UTR) with significant sequencehomology to Mycoplasma hyopneumoniae 23S ribosomal RNA juxtaposed to asequence that encodes a truncated and mutated human elongation factor 1α(Trun-EF). Stable expression of a near full-length 1.9 kb PTI-1 gene,but not the separate PTI-1 5′ UTR or Trun-EF region, in normal ratembryo fibroblast cells, CREF-Trans 6, induces an aggressive tumorigenicphenotype in athymic nude mice. Blocking PTI-1 expression with antisensePTI-1 results in reversion of transformed PTI-1 expressing cells to amore normal cellular morphology with suppression in both anchorageindependent growth and tumorigenic potential in athymic nude mice. Thesefindings document that PTI-1 is indeed an oncogene and directly blockingPTI-1 expression can nullify cancer phenotypes. In these contexts, PTI-1not only represents a gene with discriminating diagnostic properties butit may also serve as a novel target for the gene-based therapy of humanprostate and other cancers.

Prostate cancer is the most frequently diagnosed internal cancer of menin the United States and the second leading reason for cancer-relatedmale deaths (1-3). These statistics underscore the need for improvedmolecular staging of and therapeutic approaches for this prevalentneoplastic disease. Current procedures for detecting prostate cancerrely on physical examinations, monitoring serum prostate-specificantigen (PSA) levels, ultrasound, bone scans and tissue biopsy (1-3).Recent studies indicate that RT-PCR approaches using PSA specificprimers and RNA isolated from blood may provide an early indicator ofprostate cancer progression (4,5). However, all of these strategies arelimited in both their sensitivity and specificity. In addition, they donot provide the discriminatory power necessary to distinguish betweencancers that will remain localized and pose no imminent health threatand aggressive cancers resulting in progressive disease culminating inmetastasis and death.

A rapid expression cloning strategy coupled with differential RNAdisplay, screening of a human LNCaP cDNA expression library and therapid amplification of cDNA ends (RACE) approaches identified a novelputative prostate carcinoma tumor inducing oncogene, PTI-1 (6-8).Expression of PTI-1 occurs in human prostate, breast, colon and lungcarcinoma cell lines and patient-derived prostate carcinoma tissues, butnot in normal prostate or benign hypertrophic prostate (BPH) tissues(7,8). The full-length PTI-1 cDNA is 2123 bp consisting of a unique630-bp 5′ UTR with significant homology to Mycoplasma hyopneumoniae 23Sribosomal RNA fused to a sequence that is a truncated and mutated humanEF-1α (Trun-EF) (7). PCR with human genomic DNAs from normal human brainand kidneys, using PTI-1 specific 5′ UTR primers, provides evidence thatthis novel sequence is present in the human genome (8). Support for thisconclusion comes from Southern blotting of genomic DNAs. Moreover,RT-PCR, using one primer specific for the 5′ UTR and the other for theEF-1α coding region, amplifies PTI-1 transcripts from total RNAs ofprostate, breast and colon carcinoma cell lines and blood samples frompatients with metastatic prostate cancer (8). Taken together these datasuggest that the identification of PTI-1 was unlikely due to acontamination of samples with bacteria or cloning artifacts.Serial-dilution experiments indicate that PTI-1 can detect 1 prostatecarcinoma cell in 10⁸ cells not expressing PTI-1 (8). The exquisitesensitivity of PTI-1 in detecting carcinoma cells in the bloodstream ofpatients with metastatic prostate cancer, suggests that this gene willprove extremely valuable as a sensitive and specific monitor of prostatecancer progression as reflected by the presence of cancer cells in apatient's bloodstream.

The objective of the present study was to resolve if PTI-1 expressionsimply correlates with or actually controls neoplastic transformation inthe CREF-Trans 6 cell line. To define the role of PTI-1 in elicitingtransformation of CREF-Trans 6 cells, both ectopic sense (S) expressionand AS strategies were used. Expression constructs were produced thatresult in S or AS expression of specific components of the PTI-1 gene,i.e., the 5′ UTR, Trun-EF and the 1.9 kb region of PTI-1 (including partof the 5′ UTR and the Trun-EF). Pooled PTI-1 S expressing CREF-Trans 6cells are tumorigenic in nude mice, whereas no tumors form when pooled5′ UTR or Trun-EF expressing CREF-Trans 6 cells are injectedsubcutaneously into nude mice. Transient transfection assays demonstratethat only the complete PTI-1 AS construct can inhibit colony formationin PTI-1 expressing cells, including LNCaP DNA transfected nude mousetumor-derived CREF-Trans 6:4 NMT and human DU-145 cells. Stable PTI-1 ASexpression in CREF-Trans 6:4 NMT cells results in a reversion inmorphology to that of untransformed CREF-Trans 6 cells, an eliminationof PTI-1 sense RNA, a reduction in anchorage-independence and asuppression in oncogenic potential in athymic nude mice. These resultsprovide definitive evidence that PTI-1 is an oncogene and its expressionis directly involved in controlling growth and maintaining thetransformed phenotype. On the basis of the restricted expression ofPTI-1 to carcinoma cells and the ability of AS molecules to directlyinhibit expression of PTI-1 and the neoplastic phenotype, interventionin PTI-1 expression may represent a novel and effective approach for thetherapy of human prostate and other PTI-1 expressing cancers.

Materials and Methods

Cell Lines, Culture Conditions and Anchorage-Independence Assays. TheCREF-Trans 6 cell line and nude mouse tumor-derived CREF-Trans 6 cellstransfected with LNCaP DNA, CREF-Trans 6:4 NMT, have been describedpreviously (6). Human cell lines used in this study include: prostatecarcinoma (DU-145 and LNCaP), breast carcinoma (MCF-7 and T47D) andcolon carcinoma (SW480 and WiDr) (7,8). Rodent cells were grown inDulbecco's modified Eagle's medium supplemented with 5% fetal bovineserum (DMEM-5) at 37° C. in a 95% air/5% CO₂-humidified incubator. Humancells were grown in DMEM supplemented with 10% fetal bovine serum(DMEM-10). Early passage (<5) normal human prostate epithelial (NHPE)cells were obtained from Clonetics Inc., CA and grown in serum-freemedium supplied by the manufacturer. All cell lines used in the presentstudy were tested for Mycoplasma contamination using the GenProbeMycoplasma test kit (Gaithersburg, Md.) and found to be Mycoplasma free.

Expression vector constructs and DNA transfection assays. A 1.9 kb PTI-1cDNA, containing an ˜500-bp region from the 5′ UTR, the Trun-EF codingregion and the 3′ UTR, was cloned in a sense (S) and antisense (AS)orientation into a pZeoSV vector as previously described (9,10).Additionally, a 500-bp region of the 5′ UTR of PTI-1 and the Trun-EF ofPTI-1 were also cloned in a S and AS orientation into a pZeoSV vector.To study the effects of these constructs on monolayer colony formationthe vector (pZeoSV) containing no insert, PTI-1 S, PTI-1 AS, 5′ UTR S,5′ UTR AS, Trun-EF S or Trun-EF AS expression constructs weretransfected into the various cell types by the lipofectin method(GIBCO/BRL) and zeocin resistant colony formation or tumorigenicpotential in nude mice was determined (10-12).

Anchorage-independence and tumorigenicity assays. Anchorage independenceassays were performed by seeding various cell densities in 0.4% Nobleagar on a 0.8% agar base layer both of which contain growth medium (13).Colonies of ≧0.1 mm in diameter were identified with a calibrated gridunder an Olympus inverted phase-contrast microscope after 21 days.Tumorigenesis assays were performed as described by injecting 1×10⁶cells subcutaneously into athymic BALB/c nude mice and monitoringanimals for tumor development 2× per week (11-13).

RNA preparation and Northern blotting, nuclear run-on and RNaseprotection assays. Total cellular RNA was isolated by theguanidinium/phenol extraction method and Northern blotting was performedas described (9,10). Fifteen μg of RNA were denatured with glyoxal/DMSOand electrophoresed in 1% agarose gels, transferred to nylon membranesand hybridized sequentially with ³²P-labeled PTI-1 5′ UTR (500-bpregion), Zeocin and GAPDH cDNA probes (9,10). Following hybridization,the filters were washed and exposed for autoradiography. Thetranscription rates of PTI-1 5′ UTR, Trun-EF, pBR322 and GAPDH inCREF-Trans 6, CREF-Trans 6:4 NMT, 4NMT-Vector cl 1, 4NMT-PTI-1-AS cl 1,4NMT-PTI-1-AS cl 8 and 4NMT-PTI-1-AS cl 10 was determined by nuclearrun-on assays as described (12). RNase protection assays were performedusing the Ambion Ribonuclease Protection Assay Kit (Ambion, Tex.).Briefly, antisense and sense RNA probes were made by in vitrotranscription and labeled with 32P-UTP, hybridized with total cellularRNA and digested with a mixture of RNase A and RNase T1. Afterelectrophoresis in 6% polyacrylamide gels and autoradiographic exposurethe protected RNA fragments appeared as distinct bands of predictedmolecular size (14,15).

Experimental Results and Discussion

PTI-1 Is a Dominant Acting Oncogene. To determine if PTI-1 has oncogenicproperties, a 1.9 kb PTI-1 clone, missing ˜215 bp from the 5′ UTR of theoriginal PTI-1 cDNA isolated from a human prostate LNCaP cDNA library,was cloned into a pZeoSV vector and transfected into CREF-Trans 6 cells.Transfectants were selected for zeocin resistance and pooled coloniesfrom 4 separate plates were each injected into 4 athymic nude mice,total 16 nude mice. Within 10 days of injection, tumors were apparent inall animals (FIG. 5). As anticipated, nude mice injected with CREF-Trans6:4 NMT cells, resulting from transfection with high molecular weightDNA from LNCaP cells and expressing PTI-1, also induced rapidly growingtumors (FIG. 5). Seven independent tumors derived from animals injectedwith pooled PTI-1 transfected CREF-Trans 6 cells were excised andestablished in cell culture. All of these tumor-derived cell linesexhibited a transformed morphology and expressed PTI-1 and the Zeocingene (FIG. 6 and data not shown). No tumors developed when animals wereinjected with CREF-Trans 6 cells or CREF-Trans 6 cells transfected withan empty pZeoSV expression vector or pZeoSV expression vectorscontaining a 500 bp region of the 5′ UTR of PTI-1, the Trun-EF of PTI-1or a combination of the separated 500 bp 5′ UTR and the Trun-EF regionsof PTI-1 (FIG. 5). These results document that PTI-1 is a dominantacting oncogene and the intact gene is required to elicit a biologicaleffect.

Expression of an Intact Antisense PTI-1 Gene Inhibits Monolayer ColonyFormation in PTI-1 Expressing Cells. To scrutinize the role of PTI-1 inmaintenance of transformation and the tumorigenic phenotype, an ASapproach was used to selectively suppress expression of this gene.Transfection of PTI-1 AS (cloned in a pZeoSV vector permitting selectionin Zeocin) into PTI-1 expressing cells, including CREF-Trans 6:4 NMT andDU-145, results in a >50% suppression in monolayer colony formation,average of three independent experiments (FIG. 7). Transfection of PTI-1AS into T47D human breast carcinoma and SW480 human colon carcinomacells that express PTI-1 also inhibits colony formation by >50% (datanot shown). In contrast, only <10% inhibition in colony formation occursfollowing PTI-1 AS transfection of CREF-Trans 6 cells, that do notexpress PTI-1. Similarly, PTI-1 AS only inhibits colony formation by˜20% WiDr human colon carcinoma cells that do not contain PTI-1 mRNA(data not shown).

The PTI-1 cDNA consists of a unique 5′ UTR with sequence homology to 23SrRNA from Mycoplasma hyopneumoniae adjacent to a Trun-EF (7,8). It wasconsidered essential to determine if the effects on colony formationobserved with the 1.9 kb AS PTI-1 gene were specific for this moleculeor if a phenotypic response could also be induced with the AS 5′ UTR orAS Trun-EF regions of PTI-1. This was of particular relevance, since theTrun-EF might alter the expression or functionality of endogenous EF-1α,thereby causing a nonspecific negative effect on protein synthesis andcell growth in cells not expressing PTI-1 (16-21). To approach thisquestion, a 500 bp region of the 5′ UTR and the Trun-EF of PTI-1 weresubcloned in an AS orientation into the pZeoSV vector and transfectedinto CREF-Trans 6, CREF-Trans 6:4 NMT and DU-145 cells. In the case ofCREF-Trans 6:4 NMT and DU-145, a maximum ˜20% reduction in colonyformation, three separate experiments, occurred following transfectionwith the AS 5′ UTR or AS Trun-EF region of PTI-1 (FIG. 7). In CREF-Trans6, transfection with AS 5′ UTR or AS Trun-EF of PTI-1 inhibited colonyformation by only <10% (three independent experiments) (FIG. 7). Thesefindings illustrate that AS expression of an intact PTI-1 gene, but notan AS 5′ UTR or AS Trun-EF, can specifically suppress growth intransformed cells expressing this gene. This suggests that inactivationof the specific fusion gene product, containing the 5′ UTR and Trun-EFtranscript, is mandatory for evoking the biological response describedabove.

It is noteworthy that only AS expression of an intact PTI-1 gene, butnot an AS 5′ UTR or Trun-EF construct, can revert the transformedphenotype and suppress colony formation in transformed rodent and humancancer cells expressing PTI-1. As previously discussed, the PTI-1 5′ UTRshares significant homology with prokaryotic ribosomal RNA, and itscoding region is 97% homologous to human EF-1α. A possible explanationfor the lack of activity of the incomplete PTI-1 AS gene constructs mayinvolve competitive interactions of the AS 5′ UTR or AS Trun-EF PTI-1molecules with endogenous ribosomal and EF-1α RNA messages,respectively. This competitive interaction with endogenous transcriptswould predictably reduce the concentration of AS molecules tosubthreshold levels that are unable to modify PTI-1 activity and altercellular phenotypes. Alternatively, the differential effect of theintact PTI-1 AS versus the 5′ UTR AS and Trun-EF AS regions of PTI-1 mayreflect the complex interactions between AS and their cellular targetmolecules that are necessary for inhibiting gene expression andeliciting a biological response. This may include conformationalrequirements for the AS molecules that are mandatory for appropriateinteraction with their cognate S counterparts. There are precedentsindicating that not all AS oligonucleotides, even with comparablestructures, exhibit predicted effects on expression of their target gene(14). Experiments are in progress to define small regions of the PTI-1gene, such as the bridge region consisting of 5′ UTR and Trun-EFnucleotides, that may provide useful targets for AS applications.Further research addressing these issues will be crucial for designingappropriate AS PTI-1 molecules for cancer therapeutics.

Antisense Inhibition of PTI-1 Expression Suppresses Transformation Invitro. PTI-1 expressing CREF-Trans 6:4 NMT cells display a transformedmorphology that easily distinguishes them from untransformed CREF-Trans6 cells (FIG. 1). Transfection of these cells with an intact AS PTI-1results in the formation of colonies morphologically resemblinguntransformed CREF-Trans 6 (FIG. 1). This morphology change is notapparent in CREF-Trans 6:4 NMT cells transfected with the pZeoSV vector(FIG. 1). Similarly, the morphology of CREF-Trans 6:4 NMT cells isunaltered following transfection with an AS 500 bp PTI-1 5′ UTR, an ASPTI-1 Trun-EF or a combination of the separate AS 500 bp PTI-1 5′ UTRand an AS PTI-1 Trun-EF (data not shown).

Studies were performed to determine if the morphological reversion of ASPTI-1 expressing CREF-Trans 6:4 NMT cells correlates with specificchanges in cellular phenotype. Eleven morphologically-reverted Zeocinresistant colonies of CREF-Trans 6:4 NMT cells transfected with AS PTI-1were isolated and maintained as independent clonal cell lines. Themajority of clones, 7 of 11, retained their CREF-Trans 6-like morphologyeven after repeated subculture (>20 passages). When tested for anchorageindependent growth, CREF-Trans 6:4 NMT and six independent CREF-Trans6:4 NMT pZeoSV vector transformed clones grew in agar with an ˜40%efficiency (FIG. 2 and data not shown). In contrast, the majority of ASPTI-1 transfected CREF-Trans 6 clones exhibited a reduction in agargrowth to <10% (FIG. 2). Repeated passage in monolayer culture of twooriginally CREF-Trans 6-like PTI-1 transfected CREF-Trans 6:4 NMTclones, 4NMT-PTI-1-AS cl 2 and cl 5, resulted in reappearance of cellswith transformed morphology and these cells grew in agar with anintermediate or a similar efficiency as CREF-Trans 6:4 NMT andCREF-Trans 6:4 NMT pZeoSV vector transformed clones (FIG. 2). RNaseprotection assays document that 4NMT-PTI-1-AS cl 2 and cl 5 cells do notcontain PTI-1 AS mRNA (data not shown). These results confirm thatstable expression of AS PTI-1 can induce a reversion in the transformedproperties of CREF-Trans 6:4 NMT cells as established by alteredmorphology and suppression of anchorage-independence.

Stable Expression of Antisense PTI-1 Inhibits Oncogenesis. On the basisof the in vitro suppression of transformation by AS PTI-1, studies wereperformed to determine if stable AS PTI-1 expression in CREF-Trans 6:4NMT modifies oncogenic potential. CREF-Trans 6:4 NMT cells form rapidlygrowing tumors when injected subcutaneously into athymic nude mice (FIG.3A). Transfection with the pZeoSV vector does not alter the tumorigenicpotential of CREF-Trans 6:4 NMT cells. In contrast, AS PTI-1 transformedCREF-Trans 6:4 NMT cl 1, cl 6, cl 8 and cl 10, display a dramaticinhibition in tumor formation. In all cases, the majority of animalsinoculated with the AS construct remained tumor-free during the courseof the study, a minimum of 60 days. These results illustrate that ASPTI-1 also suppresses the oncogenic phenotype in vivo. In this context,gene targeting strategies using AS PTI-1 may prove amenable for thetherapy of prostate and other cancers.

Mechanism by Which Antisense PTI-1 Reverses Cancer Phenotypes. Theability of AS PTI-1 to alter the phenotype of CREF-Trans 6:4 NMT cellscould result from a specific effect of the AS on PTI-1 expression or itcould involve a trivial non-specific effect occurring independent ofalterations in PTI-1 RNA. To confirm that AS PTI-1 is expressed and itis altering PTI-1 RNA levels in morphologically reverted CREF-Trans 6:4NMT cells, RNase protection assays were conducted (FIG. 8). A PTI-1sense transcript of 357 nt and a PTI-1 AS transcript of 465 nt wassynthesized and the ability of these probes to protect in vivo producedRNA species was determined (14,15). In the case of the 357 nt PTI-1sense transcript, protection is only observed in the AS PTI-1 expressingCREF-Trans 6:4 NMT clones, i.e., 4NMT-PTI-1-AS cl 1, cl 8 and cl 10(FIG. 8). As anticipated the 357 nt protected fragment was not apparentin CREF-Trans 6:4 NMT, 4NMT-Vector cl 1, LNCaP, DU-145 or CREF-Trans 6cells lacking the AS PTI-1 transcripts. When the RNase protection assaywas performed using a 465 nt AS transcript, protection is observed inLNCaP, DU-145 and CREF-Trans 6:4 NMT cells that contain PTI-1 sensetranscripts. Absence of PTI-1 RNA results in no protection of the 465 ntAS probe. As predicted, the 465 nt AS protected band is not present inthe three PTI-1 AS expressing CREF-Trans 6:4 NMT clones or in controlCREF-Trans 6 cells. These results establish that expression of AS PTI-1in morphologically reverted CREF-Trans 6:4 NMT cells correlates withabsence of the PTI-1 message.

AS inhibition of gene expression can work at multiple levels, includingaltering transcription of a target gene, facilitating the degradation oftargeted double-stranded mRNA and/or inhibiting binding of the mRNA tothe ribosome preventing translation into protein (22-24). Asdemonstrated using RNase protection assays, AS PTI-1 prevents theappearance of PTI-1 mRNA in reverted clones. To determine if the stableexpression of AS PTI-1 can also modify transcription of the PTI-1 genenuclear run-on assays were performed (FIG. 9). The relative rate of RNAtranscription from the 5′ UTR of PTI-1 is inhibited ˜2 to 3-fold in ASPTI-1 expressing CREF-Trans 6:4 NMT cl 1, cl 8 and cl 10 in comparisonwith vector-transformed and parental CREF-Trans 6:4 NMT cells. Notranscription of the 5′ UTR is detected in CREF-Trans 6 cells. A similar˜2 to 3-fold reduction in transcription is also apparent in the AS PTI-1expressing clones when hybridized with the Trun-EF gene sequence (FIG.9). Because of high sequence homology between the Trun-EF of PTI-1 andendogenous rat EF-1α (˜91 to 94% homologous with different rat species)cross-hybridization can be anticipated. Therefore, the apparent decreasein transcription of the Trun-EF region of PTI-1 does not definitivelyprove that transcription of this gene is suppressed in these cells.These results confirm that only small changes in the transcription ofPTI-1 occur in the AS PTI-1 expressing cells, suggesting that thepredominant effect of AS PTI-1 is on steady-state mRNA levels. Thesechanges could include alterations in tanscriptional initiation,transcriptional attenuation and/or message stability.

Proposed Hypothetical Model of Action of PTI-1 as an Oncogene. Cancer isa progressive disease characterized by the appearance of new traits orthe further elaboration of existing transformation related properties inthe evolving tumor cells (25-27). Recent data provide compellingevidence for a potential link between alterations in the translationalmachinery of cells, including both eukaryotic initiation and elongationfactors, and oncogenesis (28,29). Overexpression of the eukaryoticprotein synthesis initiation factor, eIF-4E, can profoundly affectcellular physiology, including cooperating with viral oncogenes (such asv-myc and adenovirus E1A) to transform primary rodent cells (30),eliciting a tumorigenic phenotype in established rodent cells (31) andcooperating with MAX to produce a tumorigenic and metastatic phenotypein Chinese hamster ovary (CHO) cells (32). Elevated expression of EF1α,that normally functions to insure proper codon-anticodon bindinginteractions at the A site of the ribosome (28,29), also modifiescellular properties rendering both mouse and Syrian hamster cellssusceptible to carcinogen- and ultraviolet light-induced transformation(33). Enhanced levels of EF-1α are also found in tumors of the pancreas,colon, breast, lung, and stomach relative to adjacent normal tissue(34). Moreover, the data in the present paper provides directexperimental support for an association between the expression of atruncated and mutated EF1α, encoded by PTI-1, and expression of cancerphenotypes.

On the basis of studies in bacteria (elongation factor-Tu) and yeast(EF-1α), a model of action of EF-Tu/EF-1α has been proposed (16-21,35).These molecules are perceived to mediate the process of kinetic proteinproofreading that controls appropriate codon-anticodon bindinginteractions (16). Specific mutations in EF-Tu elicit dominant-negativeinhibition of protein synthesis and increase missense error rates inbacteria (16-19). Similarly, mutations (specific amino acid and alteredexpression) in EF-1α in yeast directly affect frequencies offrameshifting and amino acid misincorporations, proofreading ofcodon-anticodon interactions and suppression of nonsense mutations(20,21). These findings support a potential hypothesis for the action ofPTI-1 that involves a process we have termed “translational infidelity”(7). In this model, PTI-1 modifies EF-1α activity to introduce distinctamino acid mistakes generating mutant transforming proteins and/orpreventing cancer cells from correcting specific protein mutations thatpromote cancer progression (7). Studies are now being conducted usingSaccharomyces cerevisiae and genetically engineered mammalian cells asexperimental model systems to resolve the role of PTI-1 in regulatingprotein translation and cellular phenotype.

The mechanism by which PTI-1 functions as an oncogene requiresclarification. However, this observation provides additional support foran association between protein translational control and the neoplasticprocess (7,28-34). As discussed above, on the basis of structure, PTI-1may mediate carcinoma formation from epithelial precursor cells bymodifying normal EF-1α function in a dominant-negative manner, thereby,resulting in decreased protein translational fidelity and an inabilityto repress specific mutations in cancer cells (28,29). If the“translational infidelity” hypothesis is proven accurate, PTI-1 mayrepresent a novel class of genes that can directly effect “genomicstability” and function as an important contributor to the mutatorphenotype of cancer cells and tumor progression by altering the accuracyof protein translation. At present this possibility is onlyhypothetical. The PTI-1 oncogene may have developed as a consequence ofspecific mutations in EF-1α including a fusion with 5′ UTR sequenceswith homology to bacterial 23S ribosomal RNA (7,8). Since the unique 5′UTR of PTI-1 is found in the genomes of both normal and cancer cells, itwill be important to isolate and compare the genomic structure of thesegenes. An evaluation and analysis of these genetic elements shouldprovide important insights into the potential origin and role of PTI-1in cancer.

Conclusion. The PTI-1 gene represents a significant advance inmonitoring prostate carcinoma progression as indicated by the occurrenceof prostate carcinoma cells in a patients' circulatory system (8). Wepresently provide compelling evidence that the PTI-1 gene is adominant-acting oncogene and it can serve as a direct target forintervening in the cancer phenotype. On the basis of structure, i.e.,encoding a truncated and mutated human EF-1α, the PTI-1 gene representsthe first member of a new class of oncogenes. These data provide supportfor the hypothesis that PTI-1 is a functionally relevant geneticcomponent of prostate (and possibly breast, colon and lung) cancerdevelopment and progression and targeting this gene for inactivation mayrepresent a novel strategy for intervening in the cancer process.

References of the Fifth Series of Experiments

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Previous studies demonstrate that an antisense gene construct targetingthe majority (1.9 kb) of the PTI-1 molecule results in a suppression ofoncogenic phenotype. In contrast, antisense directed toward only the 5′UTR or the trucated EF-1α region of PTI-1 does not elicit thisphenotype. Three results argue that antisense inhibition of cancerphenotypes requires antisense molecules directed toward both the 5′ UTRand the EF-1α region of PTI-1. To test this hypothesis antisensephosphorothioate oligonucleotides were constructed based on the sequenceof the bridge specific probe (Sun, Y., Lin, J., Katz, A. E. & Fisher, P.B. (1997) Cancer Res. 57, 18-23.) region of PTI-1 (5′AAATTAAGCTATGCAGTCGG3′), BSP-AS. When CREF-trans 6 cells transformed byLNCaP human prostate cancer DNA and expressing the PTI-1 gene or DU-145hormone refractive human prostate cancer cells are seeded in agar in thepresence of as little as 2 μM of BSP-As both the size and number ofcolonies forming are decreased. The effect of the BSP-AS isdose-dependent resulting in very few colonies formed when cells aregrown in ≧50 μM of this antisense oligonucleotide. These results providedirect evidence for the potential use of the BSP-AS for inhibitingtransformation-related properties in tumor cells expressing PTI-1.

1-26 (canceled)
 27. A method for inhibiting the expression of a prostatetumor inducing gene product in a cell comprising introducing into saidcell an antisense nucleic acid molecule capable of specificallyrecognizing a prostate tumor inducing gene under conditions whereinexpression of the prostate tumor inducing gene product is inhibited. 28.A method for inhibiting the expression of a prostate tumor inducing geneproduct in a cell comprising introducing into said cell an expressionvector capable of expressing an antisense nucleic acid molecule capableof specifically recognizing a prostate tumor inducing gene underconditions wherein expression of the prostate tumor inducing geneproduct is inhibited.
 29. A method for reversing the cancer phenotype ofa cancer cell comprising introducing into said cell an antisense nucleicacid molecule capable of specifically recognizing a prostate tumorinducing gene under conditions wherein expression of the prostate tumorinducing gene product is inhibited thereby resulting in a reversal ofthe cancer phenotype.
 30. A method for reversing the cancer phenotype ofa cancer cell comprising introducing into said cell a vector capable ofexpressing a nucleic acid molecule capable of specifically recognizing aprostate tumor inducing gene wherein expression of the prostate tumorinducing gene product is inhibit thereby resulting in a reversal of thecancer phenotype.
 31. The method of claim 27-30 wherein the prostatetumor inducing gene is PTI-1.
 32. The method of claim 30 wherein theantisense nucleic acid molecule is complementary to the untranslatedregion of said prostate tumor inducing gene.
 33. The method of claim 32wherein the anti-sense nucleic acid molecule is complementary to the 5′untranslated region and the EF-1α region of the prostate tumor inducinggene.